This invention is directed to a process for immunochemically detecting uterine endometrial cancer cells in a uterine endometrial cell or endometrial tissue specimen.
Uterine endometrial cancer is increasing in Japan with westernization of eating habits and prolongation of people's lives. Today, endometrial cancer accounts for 10 to 20% of all uterine cancers. In consideration of such conditions, an item directed to a medical examination of the uterine endometrial cancer was included in the Law of Health and Medical Service System for the Aged in Jun. 1987 to spread the medical examination of the uterine endometrial cancer by law so that the cancer may be detected at an early stage.
Today, the medical examination of the uterine endometrial cancer is mainly carried out by means of cytodiagnosis, which has proved to be quite useful. The cytodiagnosis, however, requires a high skill. In addition, since most of the uterine endometrial cancer is an adenocarcinoma, discrimination between benign and malignant cell specimens on the bases of conventional morphological criteria has often been quite difficult. Therefore, there is a strong demand for a convenient, accurate process which is capable of handling a large number of specimens in a short period so that the process may be used for group examinations and mass screenings.
Immunohistochemical staining by utilizing a highly specific monoclonal antibody is a widely employed technique, and use of such a monoclonal antibody which specifically reacts with a tumor cell has been considered to be quite useful for accurately discriminating between benign and malignant cell specimens. In view of such conditions, Nozawa et al. produced a monoclonal antibody designated MSN-1, which is highly reactive with the uterine endometrial cancer cell (see Nozawa et al., Acta Obst. Gynaec. Jpn., 39, 559-566, 1987). This monoclonal antibody was applied to immunohistochemical staining of tissue specimens collected from uterine cavity of both normal people and patients suffering from the endometrial cancer, whereby more than 90% of the specimens collected from the endometrial cancer patients turned out to be positive while the normal specimens were scarcely stained (see Nozawa et al., American Journal of Obsterics and Gynecology, 161, 1079-1086, 1989). Such an immunohistochemical staining, however, depends on subjective criteria, and the results are often determined as false positive. Development of an improved detection method is therefore desired.
Further investigation of the above mentioned monoclonal antibody, MSN-1, indicated that the monoclonal antibody recognizes a sugar chain of a neutral glycolipid in the endometrial cancer cell, and that the reaction site is located at least on cell surface. In consideration of such an indication, the monoclonal antibody, MSN-1 was applied to flow cytometry, whereby the possibility of differentiating the endometrial normal cells from the cancer cells was indicated. Since the data provided by the flow cytometry is highly objective, it was concluded that the diagnosis of the uterine endometrial cancer by means of the flow cytometry would be useful (Nozawa et al., American Journal of Obsterics and Gynecology, 161, 1079-1086, 1989, supra). This method, however, requires complicated techniques as well as expensive equipment. Also, this method is incapable of examining a large number of specimens in a short period.
In addition to the problems as described above which are specific to the diagnosis of the uterine endometrial cancer, immunochemical detection generally suffers from the problem as described below.
In general, when a target substance such as a tumor marker in blood is measured and the result is determined whether it is positive or negative, namely, whether the measurement is within a normal range or not, a cut off value or a reference value is preliminarily set, and the results are determined by referring to such a value. However there are some false positive cases, wherein the tumor marker concentration in blood is high even though the sample has been collected from a normal volunteer or a patient suffering from a benign disease, and wherein the high measurement may not necessarily indicate a cancer. Various factors are estimated to be the causes of such a phenomenon including deficiency of the specificity of the antibody used and nonspecific reaction due to interfering components in the specimen.
This was also found to be the case with the diagnosis of the uterine endometrial cancer using the monoclonal antibody, MSN-1.
It was found that the monoclonal antibody, MSN-1 sometimes reacts with normal endometrial cells as well as endometrial cells of a benign endometrial disease, although the degree of the reaction was weak. Also, the labelled substance used is expected to adsorb nonspecifically. As a consequence, there were found some rare cases wherein the signal intensity indicated by the labelling agent of the labelled substance was high in spite of the absence of the endometrial cancer cell.
The cut off value is generally determined either statistically on the basis of distribution of the measurements of normal, benign disease and cancer cases, or in accordance with clinical utility.
A value equivalent to a cut off value would also be necessary for the detection of the endometrial cancer cell from endometrial cells collected from the uterine cavity. It is, however, quite difficult to collect a constant volume of the endometrial cells or tissue from the uterine cavity, and consequently, the quantity of the cells or tissue which is examined would be quite different from sample to sample. Therefore, the cut off value cannot be simply set on the basis of the signal intensity indicated by the labelling agent of the labelled substance.
This invention was developed in consideration of such a state of the art, and therefore, an object of the present invention is to provide an improved process for immunochemically detecting a human endometrial cancer cell by utilizing a monoclonal antibody which has a highly specific immunoreactivity to the endometrial cancer cell, and by simultaneously measuring two substances in the specimen and relatively evaluating the thus obtained measurements. Another object of the present invention is to provide a process which is capable conveniently, objectively, and accurately of determining the presence or the absence of the uterine endometrial cancer cell in the specimen.
In consideration of the problems associated with the diagnosis of the uterine endometrial cancer as mentioned above, the inventors of the present invention have made an intensive study to overcome such problems and developed a process by which any one can conveniently, accurately, and objectively detect endometrial cancer cell without requiring any special skill or equipment. In particular, the inventors attempted to more adequately set the cut off value.
First, the inventors decided to employ a monoclonal antibody which has a high reactivity to the uterine endometrial cancer cell, such as antibody, MSN-1, for the detection of the endometrial cancer cell, and to allow for the monoclonal antibody to react with the uterine endometrial cell specimen examined, which is immobilized on a carrier. It was also decided to use a known labelling agent for the detection.
Secondly, since an adequate setting of the cut off value is critical for more accurately determining the result of the cytodiagnosis, a means for setting the cut off value was investigated.
More illustratively, the inventors attempted to determine the total number of the cells (or an index for the total cell number) in the specimen, and to determine the ratio of number of the cells (or an index for the cell number) detected by using the antibody against the endometrial cancer cell per unit number of the cells, and then, set the cut off value on the basis of the thus determined ratio.
Typical methods for directly measuring the total number of the cells in the specimen include use of a hemocytometer and Coulter counter. It is, however, difficult to adopt such methods for the diagnosis of the uterine endometrial cancer since the number of the cells in the specimen collected from the uterine cavity is quite small.
It is also difficult to use protein content in disrupted or solubilized cells as an index for the total number of the cells in the specimen since quantitative analysis of the protein is complicated. Consequently, a more convenient process would be required for a group examination and mass screening wherein a large number of specimens must be quickly handled.
In view of such conditions, the inventors of the present invention decided to use quantity a substance which is generally present in human cells in a substantially constant amount as an index corresponding to the cell number or the quantity of the disrupted cells in the specimen, and carried out a quantitative measurement of such a substance by means of an immunochemical analysis similar to the direct detection of the uterine endometrial cancer cells. It was then confirmed that the quantity of the substance which is generally present in human cells in a substantially constant amount may be used as an index corresponding to the cell number or the quantity of the cell lysate in the specimen.
On the bases of the above-described findings, it was then found out that the presence or the absence of the uterine endometrial cancer cell in the specimen may be determined by, in the first place, immunochemically measuring a value corresponding to the quantity of the endometrial cancer cell in the specimen using the monoclonal antibody such as the antibody, MSN-1 which is highly reactive to the uterine endometrial cancer cell to measure the quantity of antigen which binds to such an antibody, as well as a value corresponding to the quantity of the substance which is generally present in human cells in a substantially constant amount, and in the next place, either simply comparing the thus obtained values, or calculating a ratio of the values and evaluating the ratio by referring to a preliminarily set cut off value to determine the presence or the absence of the uterine endometrial cancer cell in the specimen. It was also found that, by using this process, any one can conveniently, objectively, and accurately detect the uterine endometrial cancer cell without requiring any special technique or equipments.
The present invention has been completed on the bases of such findings.
According to first aspect of the present invention, there is provided a process for detecting a human uterine endometrial cancer cell by using a monoclonal antibody having a highly specific immunoreactivity to the human uterine endometrial cancer cell, comprising the steps of
(a) preparing a specimen by collecting endometrial cells and/or endometrial tissue from the cavity of the uterus, and optionally disrupting the collected cells and/or the tissue,
(b) immobilizing a predetermined amount of said specimen on two or more reaction areas on one or more carriers,
(c) adding a labelled monoclonal antibody prepared by binding a labelling agent either directly or indirectly to said monoclonal antibody, and a labelled substance B prepared by binding a labelling agent either directly or indirectly to a substance B which is reactive to a substance A which is generally present in human cells, to different areas of said reaction areas onto which said specimen had been immobilized, respectively, to allow for said immobilized specimen to react with said labelled monoclonal antibody and with said labelled substance B, respectively,
(d) washing said carrier to remove said labelled monoclonal antibody and said labelled substance B which failed to bind to said immobilized specimen,
(e) measuring signal intensities y and x of said labelling agents of said labelled monoclonal antibody and said labelled substance B, which had been bound to said immobilized specimen, respectively, and
(f) determining the presence or absence of the uterine endometrial cancer cell in the specimen by evaluating the signal intensity y indicated by the labelling agent of said labelled monoclonal antibody in relation to the signal intensity x indicated by the labelling agent of said labelled substance B.
This method may be referred to as Assay method I in the following description.
According to second aspect of the present invention, there is provided a process for detecting a human uterine endometrial cancer cell by using a monoclonal antibody having a highly specific immunoreactivity to the human uterine endometrial cancer cell, comprising the steps of
(a) preparing a specimen by collecting endometrial cells and/or endometrial tissue from the cavity of the uterus, and optionally disrupting the collected cells and/or the tissue,
(b) immobilizing a predetermined amount of said specimen on two or more reaction areas on one or more carriers,
(c) adding said monoclonal antibody and a substance B which is reactive to a substance A which is generally present in human cells to different areas of said reaction areas onto which said specimen had been immobilized to allow for said immobilized specimen to react with said monoclonal antibody and with said substance B, respectively,
(d) adding a labelled substance C prepared by binding a labelling agent either directly or indirectly to a substance C which reacts with said monoclonal antibody, and a labelled substance D prepared by binding a labelling agent either directly or indirectly to a substance D which reacts with said substance B, to said different reaction areas, respectively, to allow for said monoclonal antibody and said substance B to react with said labelled substance C and said labelled substance D, respectively,
(e) washing said carrier to remove said monoclonal antibody, said substance B, said labelled substance C, and said labelled substance D, which failed to directly or indirectly bind to said immobilized specimen,
(f) measuring signal intensities y and x of said labelling agents of said labelled substance C and said labelled substance D, which had been bound to said immobilized specimen, respectively, and
(g) determining the presence or absence of the uterine endometrial cancer cell in the specimen by evaluating the signal intensity y indicated by the labelling agent of said labelled substance C in relation to the signal intensity x indicated by the labelling agent of said labelled substance D.
This method may be referred to as Assay method II in the following description.